**4.2 Anti-QS activity of natural furanones**

Among the widely studied natural furanones, (5Z)-4-bromo-5- (bromomethylene)-3-butyl-2(5H)-furanone and Furanone 4-hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF), Furanone F202 show strong anti-biofilm activity by up to 55%. Based on the study of Ren et al. [22], a natural furanone, known as (5Z)-4 bromo-5- (bromomethylene)-3-butyl-2(5H)-furanone was demonstrated to attenuate biofilm formation in *E. coli*, reducing average biofilm thickness by 55%. Moreover, lower furanone concentrations (64.5 μM) significantly decreased *E. coli* swarming motility. On the other hand, investigations by Witsø et al. [23] into the impact of synthetic brominated furanones demonstrated that these compounds could also decrease *E. coli* biofilm wall thickness and surface area coverage by up to 50%. Brominated furanones, added at 50 μM, could suppress swarming motility and lower biofilm production by up to 40% in the foodborne pathogen *E. coli* 0103:H2. These important works [23] clearly demonstrated that natural furanones can interfere with QS processes and that the phenomenon could be used to combat virulence in human pathogens. Moreover, the study of Choi et al. [24] proved that natural furanones greatly reduce the production of *P. aeruginosa* virulence factors, including protease (up to 43%), chitinase and pyoverdine by almost 100% (**Table 2**) [24].



**Table 2.**

*List of natural furanone inhibitors.*

### **4.3 Anti-QS activity of synthetic furanones**

The process of developing synthetic furanones began in the 1980s and it usually starts with relative simple compounds, such as dimethyl ketones and acetals or other straight forward organic precursors. Then, it is also possible to modify existing furanone compounds and add existing furanone structures, as it is highlighted in **Table 3**. Recently, the majority of research on furanones-mediated QS inhibitors has been conducted on the effects of these compounds on human pathogens, especially on the


*Efficacy of Natural and Synthetic Biofilm Inhibitors Associated with Antibiotics… DOI: http://dx.doi.org/10.5772/intechopen.112408*


#### **Table 3.**

*List of synthetic furanone inhibitors.*

model organisms *E. coli* and *P. aeruginosa* [20]. It was the synthesis of a range of structurally diverse bromine-, chlorine and iodine-containing furanones using a variety of palladium-catalysed coupling reactions was recently described [1]. The finding of this study [1] is interesting, as furanone is an ideal QS disruptor. Various compounds from the furanone library were screened for their inhibitory effects on biofilm production in opportunistic human pathogens and were found to potently suppress bacterial biofilm formation in *S. enterica*, *S. aureus*, *P. aeruginosa*, and, to a lesser extent, *E. coli*. Compounds which inhibited biofilm formation do not generally impact bacterial growth, highlighting their potential as QS inhibitors. According to the Furanone Library [1], tribromofuranone was found to be the most active compound decreasing biofilm formation in *S. enterica* by 72% and *S. aureus* by 71% at a concentration of 50 μM, whereas methyl-substituted dibromofuranone was the most potent inhibitor, which reduces biofilm growth *P. aeruginosa* PAR7244 by 44% compared to a 70% reduction in PAO1. For *E. coli* biofilm, bis-4-methoxyphenylacetylene was the most active compound, which inhibits *E. coli* ATCC9637 biofilm growth by 31%. Moreover, it was tested whether synthesized furanones, with relevant anti-biofilm activity, were able to disturb mixed fungal-bacterial biofilms. It was confirmed that the chosen bromofuranones and chloroiodofuranones were initially subjected to testing for their effect on monospecific biofilms of *P. aeruginosa* and *C. albicans* with confocal laser scanning microscopy (CLSM). Thus, it was found that all of them decrease the biomass of both microorganisms within the mixed biofilms [1].

### **5. Anti-biofilm activity of natural compounds**

Natural products exhibit higher structural and biochemical variety than synthetic compounds, making them useful for the advancement of anti-biofilm agents [27]. More recently, there have been reviews of bacterial products that include small molecules, enzymes, exopolysaccharides and isolated peptides displaying anti-biofilm activities toward different pathogens [28]. Moreover, several studies have demonstrated solid evidences that plants [29] and marine-derived products [15] are an excellent source to provide abundant natural compounds for the development of preventative and therapeutic agents against biofilm-based infections (**Table 1**).

#### **5.1 Anti-biofilm activity of marine and bacterial-derived products**

Concerning antibiofilm activity of marine-derived products, the study by Oluwabusola et al. [15] proved that psammaplin A and bisaprasin, isolated from marine sponges, could be a potent QS inhibitory agents by preventing *P. aeruginosa* PAO1 biofilm formation. The present results indicated that psammaplin a showed moderate to significant inhibition against QS gene promoters, with IC50 values ranging from 30.69 to 2.64 μM. In contrast, bisaprasin showed significant inhibition for both biosensor strains, with equal IC50 values. Hence, using marine sources to find novel QS inhibitors as antipathogenic drugs to combat antimicrobial resistance has high potentials. Concerning antibiofilm activity of bacterial-derived products, the study of Zhang et al. (2021) described a novel and effective anti-biofilm compound named maipomycin A (MaiA), which was isolated from the metabolites of a rare actinomycete strain *Kibdelosporangium phytohabitans* XY-R10. This compound demonstrated a broad spectrum of anti-biofilm activities against Gram-negative bacteria [8].

#### **5.2 Anti-biofilm activity of thymol**

The study of Valliammai et al. [17] demonstrated the anti-biofilm potential of thymol against methicillin resistant *S. aureus* (MRSA) by inhibition of staphyloxanthin biosynthesis. The staphyloxanthin inhibitory potential of thymol was assessed against MRSA in terms of quality and quantity. It was demonstrated that 100 μg/mL concentration of thymol brings about 90% of staphyloxanthin inhibition. In addition, it was confirmed that thymol treatment makes MRSA more susceptible to reactive oxygen species. Experimental analyses were also confirmed by molecular docking analysis and in vitro measurement of metabolic intermediates of staphyloxanthin. It was also revealed that thymol could possibly interact with CrtM, which is involved in staphyloxanthin biosynthesis to inhibit production. In addition, reduction in staphyloxanthin by thymol treatment increases the membrane fluidity and makes MRSA cells more susceptible to Polymyxin B, an antibiotic targeting membrane. Thus, the present study suggests thymol as a potential alternative to antibiotics to combat MRSA infections. It can also be used as adjuvant in antimicrobial treatments [17]. Likewise, Ndezo et al. [16] showed the synergistic effect of the anti-biofilm potential of thymol and piperine either alone or combined with three aminoglycoside antibiotics were evaluated against the biofilm of *K. pneumoniae*. Their effect were also tested on either formed or pre-formed biofilms. It was found that the minimal biofilm inhibition concentration (MBIC) of streptomycin was reduced 16- to 64-fold when associated with thymol, whereas the MBIC of kanamycin was decreased 4-fold when associated with piperine. In addition, the minimal biofilm eradication concentration (MBEC) values of streptomycin, amikacin, and kanamycin were 16- to 128-fold, 4- to 128-fold, and 8- to 256-fold higher than the planktonic minimum inhibitory concentration (MIC), respectively. Therefore, thymol, in combination with antibiotics, has shown a broad synergistic activity in both inhibiting biofilm formation and destroying pre-formed biofilm of *K. pneumoniae* [16].

The synergistic effects associated with the combination of thymol or piperine along with the three considered aminoglycoside antibiotics indicate that thymol and piperine are very promising agents for the development of new antibacterial combination therapies to combat biofilm-associated infections. The study by Valliammai et al. (2020) aimed to decrypt the molecular mechanism for the anti-biofilm activity of thymol toward MRSA and to evaluate the ability of thymol to enhance the antibacterial activity of rifampicin. Thymol markedly inhibited 88% of MRSA biofilm formation at 100 μg/mL and decreased MRSA adhesion to human plasma-coated glass, stainless steel, and titanium surfaces, as demonstrated by microscopic analysis. In fact, *Efficacy of Natural and Synthetic Biofilm Inhibitors Associated with Antibiotics… DOI: http://dx.doi.org/10.5772/intechopen.112408*

thymol reinforced the antibacterial efficacy and biofilm eradication of rifampicin against MRSA and also minimized the formation of persisters. Thus, the present study suggests that thymol is a very promising combinatory agent candidate to enhance the antibacterial activity of rifampicin for persistent MRSA infections [18].

## **6. Conclusion**

Bacterial biofilms appear in many infections that are related to diverse medical implants and well defined body sites such as the urinary tract, lungs, wounds and their resistance to antimicrobial treatments is a serious problem in clinical settings. It is, therefore, imperative to study efficient solutions to this problem and to find an alternative to our current armory of antibiotics. The challenges related to biofilm infections have prompted researchers to seek a better understanding of the molecular mechanisms involved in biofilm formation, which has led to the identification of several steps in biofilm formation that could be targeted to eradicate these serious infections. Within this context, the combination of current antibiotics with potential anti-biofilm and anti-toxic agents that interfere with the QS without stimulating the incidence of resistance is a new therapeutic strategy aiming to reduce the antibiotic dosages. In this study, a screening of the most studied molecules with anti-biofilm activity, associated with or not with antibiotics, is performed. The different antibiofilm molecules investigated here have various modes of action including (i) inhibition via interference in QS pathways by 3-PPA, AS10, mBTL, natural and synthetic furanones and natural compounds, (ii) adhesion mechanism, (iii) disruption of extracellular DNA, proteins, lipopolysaccharides, exopolysaccharides and secondary messengers involved in various signaling pathways like small molecule DGCinhibitors of c-di-GMP signaling. As QS and c-di-GMP signaling govern the

production of virulence factors and some of the protective mechanisms operating in the biofilm mode, development of chemical compounds capable of preventing formation of biofilms by targeting these two major systems could be used to treat biofilmassociated infections. However, studies on the structural modifications on these molecules and their minimal effective concentration without posing harmful side effects should be made in future studies in order to improve their efficacy (**Figure 1**).
